Microdomains in Hippocampal Neurons

Ca 2 -binding proteins are ubiquitously expressed throughout the CNS and serve as valuable immunohistochemical markers for certain types of neurons. However, the functional role of most Ca 2 -binding proteins has to date remained obscure because their concentration in central neurons is not known. In this study, we investigate the intracellular concentration of the widely expressed Ca 2 -binding protein calbindin-D28k in adult hippocampal slices using patch-clamp recordings and immunohistochemistry. First, we show that calbindin-D28k freely exchanges between patch pipette and cytoplasm during whole cell patch-clamp recordings with a time constant of 10 min. Substituting known concentrations of recombinant calbindin-D28k in patch pipettes enabled us to determine the endogenous calbindin-D28k concentration by postrecording immunohistochemistry. Using this calibration procedure, we find that mature granule cells (doublecortin ) contain 40 M, and newborn granule cells (doublecortin ) contain 0 –20 M calbindin-D28k. CA3 stratum radiatum interneurons and CA1 pyramidal cells enclose 47 and 45 M calbindin-D28k , respectively. Numerical simulations showed that 40 M calbindin-D28k is capable of tuning Ca 2 microdomains associated with action potentials at the mouth of single or clustered Ca 2 channels: calbindin-D28k reduces the increment in free Ca 2 at a distance of 100 and 200 nm by 20 and 35%, respectively, and strongly accelerates the collapse of the Ca 2 gradient after cessation of Ca 2 influx. These data suggest that calbindin-D28k equips hippocampal neurons with 160 M mobile, high-affinity Ca 2 -binding sites ( S 200) that slow and reduce global Ca 2 signals while they enhance the spatiotemporal fidelity of submicroscopic Ca 2 signals.